Temporal expression profile of megakaryocytic differentiation primary CD34+ cell culture
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE3839 | GEO | 2006/11/08
SECONDARY ACCESSION(S): PRJNA104987
REPOSITORIES: GEO
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Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of Mk differentiation human CD34-positive hematopoietic stem and progenitor cells cultured with thrombopoietin, interleukin-3, and Flt3-ligand. The goal this study was to identify genes involved in the various facets of Mk differentiation including commitment, polyploidization, proplatelet formation, and apoptosis. Experiment Overall Design: G-CSF mobilized peripheral blood CD34-positive cells were cultured with TPO, IL-3, and Flt3-L to induce Mk differentiation. Samples prior to day 5, including uncultured starting cells, were analyzed directly, whereas samples after and including day 5 were positively selected for CD41a expression immediately prior to RNA isolation. Three biological replicate experiments were analyzed and approximately one-half of the samples from each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.
2008-06-12 | E-GEOD-3839 | biostudies-arrayexpress
Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of phorbol-ester-induced Mk differentiation of the megakaryoblastic CHRF-288-11 (CHRF) cell line – a model system for investigating megakaryopoiesis. The goals of this study were to (1) verify the megakaryocytic nature of the CHRF cell line at the transcriptional level, and (2) extract novel insights into the key facets of Mk maturation including polyploidization, proplatelet formation, and apoptosis. Keywords: time course
2006-11-08 | GSE3838 | GEO
Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of phorbol-ester-induced Mk differentiation of the megakaryoblastic CHRF-288-11 (CHRF) cell line â a model system for investigating megakaryopoiesis. The goals of this study were to (1) verify the megakaryocytic nature of the CHRF cell line at the transcriptional level, and (2) extract novel insights into the key facets of Mk maturation including polyploidization, proplatelet formation, and apoptosis. Experiment Overall Design: CHRF-288 cells were cultured in the presence of 10 ng/mL phorbol ester (PMA) or equivalent volume of DMSO solvent (0.02%). Unstimulated control cells from time zero (exponentially growing CHRF cells) were also analyzed. For PMA treated legs, only the adherent cells were included in the transcriptional analysis. Two biological replicate experiments were analyzed and approximately one-half of the samples with each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.
2008-06-12 | E-GEOD-3838 | biostudies-arrayexpress
Project description:Culture of hematopoietic stem and progenitor cells in the presence of thrombopoietin induces megakaryocytic differentiation. Addition of nicotinamide to thrombopoietin-stimulated cultures results in significant increases in megkaryocytic differentiation including greater polyploidization and enhanced proplatelet formation. This study focuses on understanding the differences in the temporal gene expression pattern during differentiation with and without nicotinamide. Nicotinamide was added on day 5. Keywords: treatment response
2006-08-25 | GSE4974 | GEO
Project description:Culture of hematopoietic stem and progenitor cells in the presence of thrombopoietin induces megakaryocytic differentiation. Addition of nicotinamide to thrombopoietin-stimulated cultures results in significant increases in megkaryocytic differentiation including greater polyploidization and enhanced proplatelet formation. This study focuses on understanding the differences in the temporal gene expression pattern during differentiation with and without nicotinamide. Nicotinamide was added on day 5. Experiment Overall Design: Two biological experiments were analyzed. For each experiment, a sample was taken on day 5, before nicotinamide addition, and on days 6, 8, and 10 from both nicotinamide and control treated cultures. Each sample was analyzed in duplicate for a total of 14 hybridizations per culture or 28 total hybridizations.
2009-04-08 | E-GEOD-4974 | biostudies-arrayexpress
Project description:EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and endomitosis and decreases proplatelet formation. EZH2 inhibitors similarly reduce polyploidization and proplatelet formation of JAK2V617F MK. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression for a set of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and/or CDKN2D could partially rescue the percentage of polyploid MKs. However only CDKN1A was regulated by H3K27me3 suggesting that EZH2 controls MK polyploidization through a direct regulation of CDKN1A and indirectly of CDKN2D.
2021-11-02 | E-MTAB-8609 | biostudies-arrayexpress
Project description:EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and endomitosis and decreases proplatelet formation. EZH2 inhibitors similarly reduce polyploidization and proplatelet formation of JAK2V617F MK. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression for a set of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and/or CDKN2D could partially rescue the percentage of polyploid MKs. However only CDKN1A was regulated by H3K27me3 suggesting that EZH2 controls MK polyploidization through a direct regulation of CDKN1A and indirectly of CDKN2D.
2021-03-31 | E-MTAB-8611 | biostudies-arrayexpress
Project description:EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and endomitosis and decreases proplatelet formation. EZH2 inhibitors similarly reduce polyploidization and proplatelet formation of JAK2V617F MK. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression for a set of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and/or CDKN2D could partially rescue the percentage of polyploid MKs. However only CDKN1A was regulated by H3K27me3 suggesting that EZH2 controls MK polyploidization through a direct regulation of CDKN1A and indirectly of CDKN2D.
2021-11-02 | E-MTAB-8610 | biostudies-arrayexpress
Project description:S78454 (Abexinostat, PCI-24781) is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia, whose mechanism is partially understood. We have tested its effect at doses reached in patients plasma on in vitro megakaryopoiesis derived from human CD34+ cells. When added at day 0 in culture, S78454 inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. This effect only required a short incubation period. When added later on (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet formation. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2 signaling. MK gene profiling revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. Double DNA strand breaks were increased as attested by elevated gH2AX phosphorylation level. Consequently, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, but only partially the defect in proplatelet formation. These results suggest that HDACi induces a thrombocytopenia by a p53 dependent mechanism along MK differentiation and a p53 independent mechanism for proplatelet formation.
2013-06-30 | E-MTAB-1180 | biostudies-arrayexpress
Project description:In order to examine genes whose expression is altered as a result of the knock-down (KD) of p53, we used gene expression microarray analysis based on the CHRF cell line, a human megakaryoblastic cell line, which upon PMA stimulation undergoes polyploidization, and extends proplatelet-like cytoplasmic protrusions combined with apoptosis. The CHRF cell line has been validated in our laboratory as a good model of in vitro megakaryopoiesis by comparison to hematopoietic stem cells undergoing Mk differentiation. As described, we have previously generated stable p53-KD CHRF cell lines by lentiviral delivery of microRNA-adapted-short hairpin sequences targeting p53 as well as a suitable scrambled-control cell line.
2011-07-27 | GSE30984 | GEO